Transcription factor NAC78 cooperates with NAC78 INTERACTING PROTEIN6 to confer drought tolerance in rice.

NAC (NAM, ATAF1/2, and CUC2) family transcription factors are involved in several cellular processes, including responses to drought, salinity, cold, and submergence. However, whether or how certain NAC proteins regulate drought tolerance in rice (Oryza sativa) remain unclear. In this study, we show that over-expression of OsNAC78 enhanced rice resistance to drought treatment, whereas Osnac78 mutant plants were susceptible to drought stress. We further characterized the OsNAC78 interacting protein, named NAC78 interacting protein6 (OsNACIP6), and found that it conferred rice drought tolerance. Our results demonstrate that OsNACIP6 enhanced transcription of OsNAC78 and promoted the expression of its downstream target OsGSTU37, encoding a glutathione reductase. The ABRE4 cis-element in the promoter region of OsNACIP675-1-127 conferred significant up-regulation of OsNACIP6 expression and initiated the OsNACIP6/OsNAC78-OsGSTU37 module that facilitates rice growth under drought conditions. Together, our results uncover a transcriptional module composed of OsNACIP6, OsNAC78, and OsGSTU37 and provide insights into the molecular mechanisms underlying the drought stress response in rice.

RNA-seq profiling of primary calli induced by different media and photoperiods for japonica rice 'Yunyin'.

The induction of embryogenic calli plays a vital role in the genetic transformation and regeneration of rice (Oryza sativa L.). Despite progress in rice tissue culture, the molecular mechanisms of embryogenic callus induction remain unknown. In this study, gene expression profiles associated with calli were comprehensively analyzed during callus induction of japonica rice 'Yunyin'. We first confirmed that NMB medium with 24 h of light and 0 h of dark (NMB-L) was the optimal condition for 'Yunyin' callus induction, while J3 medium with 0 h of light and 24 h of dark (J3-D) was the worst condition. After transcriptome analysis, 33,597 unigenes were assembled, among which we identified 6,063 DEGs (Differentially Expressed Genes) related to media and seven DEGs related to photoperiod. Phenylpropanoid biosynthesis, plant hormone signal, and starch and sucrose metabolism were the top three pathways affected by media, while the circadian rhythm-plant pathway was associated with photoperiod. Furthermore, we identified two candidate genes, Os01g0965900 and Os12g0555200, affected by both medium and photoperiod. Statistical analysis of RNA-seq libraries showed that the expression levels of these two genes in J3-D calli were over 2.5 times higher than those in NMB-L calli, which was further proved by RT-qPCR analysis. Based on FPKM (Fragments Per Kilobase of transcript Per Million mapped reads), unigenes belonging to the NMB-L group were mainly assigned to ribosome, carbon metabolism, biosynthesis of amino acids, protein processing in endoplasmic reticulum, and plant hormone signal transduction pathways. We transformed Os12g0555200Nip and Os12g05552009311 into 'Nipponbare' calli and observed their effects on the growth and development process of rice calli using TEM (Transmission Electron Microscopy) and SEM (Scanning Electron Microscopy). Observations showed that Os12g05552009311 was more disadvantageous to rice callus growth than Os12g0555200Nip. Our results reveal that the Os12g0555200, identified from transcriptomic profiles, has a negative influence during 'Yunyin' callus induction. Supplementary information: The online version contains supplementary material available at 10.1007/s11032-022-01283-y.

Earlier Degraded Tapetum1 (EDT1) Encodes an ATP-Citrate Lyase Required for Tapetum Programmed Cell Death.

In flowering plants, the tapetum cells in anthers undergo programmed cell death (PCD) at the late meiotic stage, providing nutrients for further development of microspores, including the formation of the pollen wall. However, the molecular basis of tapetum PCD remains elusive. Here we report a tapetum PCD-related mutant in rice (Oryza sativa), earlier degraded tapetum 1 (edt1), that shows complete pollen abortion associated with earlier-than-programmed tapetum cell death. EDT1 encodes a subunit of ATP-citrate lyase (ACL), and is specifically expressed in the tapetum of anthers. EDT1 localized in both the nucleus and the cytoplasm as observed in rice protoplast transient assays. We demonstrated that the A and B subunits of ACL interacted with each other and might function as a heteromultimer in the cytoplasm. EDT1 catalyzes the critical steps in cytosolic acetyl-CoA synthesis. Our data indicated a decrease in ATP level, energy charge, and fatty acid content in mutant edt1 anthers. In addition, the genes encoding secretory proteases or lipid transporters, and the transcription factors known to regulate PCD, were downregulated. Our results demonstrate that the timing of tapetum PCD must be tightly regulated for successful pollen development, and that EDT1 is involved in the tapetum PCD process. This study furthers our understanding of the molecular basis of pollen fertility and fecundity in rice and may also be relevant to other flowering plants.

A GARP transcription factor anther dehiscence defected 1 (OsADD1) regulates rice anther dehiscence.

Anther dehiscence, one of the essential steps in pollination and double fertilization, is regulated by a complex signaling pathway encompassing hormones and environmental factors. However, key components underlying the signaling pathway that regulate anther dehiscence remain largely elusive. Here, we isolated a rice mutant anther dehiscence defected 1 (Osadd1) that exhibited defects in anther dehiscence and glume open. Map-based cloning revealed that OsADD1 encoded a GARP (Golden2, ARR-B and Psr1) transcription factor. Sequence analysis showed that a single base deletion in Osadd1 mutant resulted in pre-termination of the GARP domain. OsADD1 was constitutively expressed in various tissues, with more abundance in the panicles. The major genes associated with anther dehiscence were affected in the Osadd1 mutant, and the expression level of the cellulose synthase-like D sub-family 4 (OsCSLD4) was significantly decreased. We demonstrate that OsADD1 regulated the expression of OsCSLD4 by binding to its promoter, and affects rice anther dehiscence.

The catalytic subunit of magnesium-protoporphyrin IX monomethyl ester cyclase forms a chloroplast complex to regulate chlorophyll biosynthesis in rice.

KEY MESSAGE: YGL8 has the dual functions in Chl biosynthesis: one as a catalytic subunit of MgPME cyclase, the other as a core component of FLU-YGL8-LCAA-POR complex in Chl biosynthesis. Magnesium-protoporphyrin IX monomethyl ester (MgPME) cyclase is an essential enzyme involved in chlorophyll (Chl) biosynthesis. However, its roles in regulating Chl biosynthesis are not fully explored. In this study, we isolated a rice mutant yellow-green leaf 8 (ygl8) that exhibited chlorosis phenotype with abnormal chloroplast development in young leaves. As the development of leaves, the chlorotic plants turned green accompanied by restorations in Chl content and chloroplast ultrastructure. Map-based cloning revealed that the ygl8 gene encodes a catalytic subunit of MgPME cyclase. The ygl8 mutation caused a conserved amino acid substitution (Asn182Ser), which was related to the alterations of Chl precursor content. YGL8 was constitutively expressed in various tissues, with more abundance in young leaves and panicles. Furthermore, we showed that expression levels of some nuclear genes associated with Chl biosynthesis were affected in both the ygl8 mutant and YGL8 RNA interference lines. By transient expression in rice protoplasts, we found that N-terminal 40 amino acid residues were enough to localize the YGL8 protein to chloroplast. In vivo experiments demonstrated a physical interaction between YGL8 and a rice chloroplast protein, low chlorophyll accumulation A (OsLCAA). Moreover, bimolecular fluorescence complementation assays revealed that YGL8 also interacted with the other two rice chloroplast proteins, viz. fluorescent (OsFLU1) and NADPH:protochlorophyllide oxidoreductase (OsPORB). These results provide new insights into the roles of YGL8, not only as a subunit with catalytic activity, but as a core component of FLU-YGL8-LCAA-POR complex required for Chl biosynthesis.

OsBBP1, a newly identified protein containing DUF630 and DUF632 domains confers drought tolerance in rice.

Domain of unknown function (DUF) protein families, which are uncharacterized and numerous within the Pfam database. Recently, studies have demonstrated that DUFs played crucial roles in plant development, but whether, or how, they function in drought resistance remain unclear. In this study, we identified the Os03g0321500 gene, encoding OsbZIP72 binding protein 1 (OsBBP1), as a target of OsbZIP72 using chromatin immunoprecipitation sequencing in rice. OsBBP1 is a novel member of DUFs, which localize both in the nuclei and cytoplasm of rice protoplasts. Furthermore, yeast one-hybrid and electrophoretic mobility shift assays confirmed the specific binding between OsbZIP72 and OsBBP1. Additionally, a luciferase reporter analysis illustrated that OsbZIP72 activated the expression of OsBBP1. Drought tolerance experiments demonstrate that the OsBBP1 CRISPER-CAS9 transgenic mutants were sensitive to drought stress, but the transgenic OsBBP1 over-expressing rice plants showed enhanced drought resistance. Moreover, drought tolerance experiments in a paddy field suggested that OsBBP1 contributed to less yield or yield-related losses under drought conditions. Mechanistically, OsBBP1 might confer drought resistance by inducing more efficient reactive oxygen species (ROS) scavenging. Several ROS scavenging-related genes showed increased expression levels in OsBBP1 overexpression lines and decreased expression levels in OsBBP1 CRISPER-CAS9 mutants under drought conditions. Thus, OsBBP1, acting downstream of OsbZIP72, contributes to drought resistance and causes less yield or yield-related losses under drought conditions.

A toxin-antidote system contributes to interspecific reproductive isolation in rice.

Breakdown of reproductive isolation facilitates flow of useful trait genes into crop plants from their wild relatives. Hybrid sterility, a major form of reproductive isolation exists between cultivated rice (Oryza sativa) and wild rice (O. meridionalis, Mer). Here, we report the cloning of qHMS1, a quantitative trait locus controlling hybrid male sterility between these two species. Like qHMS7, another locus we cloned previously, qHMS1 encodes a toxin-antidote system, but differs in the encoded proteins, their evolutionary origin, and action time point during pollen development. In plants heterozygous at qHMS1, ~ 50% of pollens carrying qHMS1-D (an allele from cultivated rice) are selectively killed. In plants heterozygous at both qHMS1 and qHMS7, ~ 75% pollens without co-presence of qHMS1-Mer and qHMS7-D are selectively killed, indicating that the antidotes function in a toxin-dependent manner. Our results indicate that different toxin-antidote systems provide stacked reproductive isolation for maintaining species identity and shed light on breakdown of hybrid male sterility.

SH3P2, an SH3 domain-containing protein that interacts with both Pib and AvrPib, suppresses effector-triggered, Pib-mediated immunity in rice.

Plants usually keep resistance (R) proteins in a static state under normal conditions to avoid autoimmunity and save energy for growth, but R proteins can be rapidly activated upon perceiving pathogen invasion. Pib, the first cloned blast disease R gene in rice, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, mediates resistance to the blast fungal (Magnaporthe oryzae) isolates carrying the avirulence gene AvrPib. However, the molecular mechanisms about how Pib recognizes AvrPib and how it is inactivated and activated remain largely unclear. In this study, through map-based cloning and CRISPR-Cas9 gene editing, we proved that Pib contributes to the blast disease resistance of rice cultivar Yunyin (YY). Furthermore, an SH3 domain-containing protein, SH3P2, was found to associate with Pib mainly at clathrin-coated vesicles in rice cells, via direct binding with the coiled-coil (CC) domain of Pib. Interestingly, overexpression of SH3P2 in YY compromised Pib-mediated resistance to M. oryzae isolates carrying AvrPib and Pib-AvrPib recognition-induced cell death. SH3P2 competitively inhibits the self-association of the Pib CC domain in vitro, suggesting that binding of SH3P2 with Pib undermines its homodimerization. Moreover, SH3P2 can also interact with AvrPib and displays higher affinity to AvrPib than to Pib, which leads to dissociation of SH3P2 from Pib in the presence of AvrPib. Taken together, our results suggest that SH3P2 functions as a "protector" to keep Pib in a static state by direct interaction during normal growth but could be triggered off by the invasion of AvrPib-carrying M. oryzae isolates. Our study reveals a new mechanism about how an NLR protein is inactivated under normal conditions but is activated upon pathogen infection.

OsMFS1/OsHOP2 Complex Participates in Rice Male and Female Development.

Meiosis plays an essential role in the production of gametes and genetic diversity of posterities. The normal double-strand break (DSB) repair is vital to homologous recombination (HR) and occurrence of DNA fragment exchange, but the underlying molecular mechanism remain elusive. Here, we characterized a completely sterile Osmfs1 (male and female sterility 1) mutant which has its pollen and embryo sacs both aborted at the reproductive stage due to severe chromosome defection. Map-based cloning revealed that the OsMFS1 encodes a meiotic coiled-coil protein, and it is responsible for DSB repairing that acts as an important cofactor to stimulate the single strand invasion. Expression pattern analyses showed the OsMFS1 was preferentially expressed in meiosis stage. Subcellular localization analysis of OsMFS1 revealed its association with the nucleus exclusively. In addition, a yeast two-hybrid (Y2H) and pull-down assay showed that OsMFS1 could physically interact with OsHOP2 protein to form a stable complex to ensure faithful homologous recombination. Taken together, our results indicated that OsMFS1 is indispensable to the normal development of anther and embryo sacs in rice.